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1.
Viruses ; 13(10)2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34696497

RESUMO

Respiratory viruses are known to be the most frequent causative mediators of lung infections in humans, bearing significant impact on the host cell signaling machinery due to their host-dependency for efficient replication. Certain cellular functions are actively induced by respiratory viruses for their own benefit. This includes metabolic pathways such as glycolysis, fatty acid synthesis (FAS) and the tricarboxylic acid (TCA) cycle, among others, which are modified during viral infections. Here, we summarize the current knowledge of metabolic pathway modifications mediated by the acute respiratory viruses respiratory syncytial virus (RSV), rhinovirus (RV), influenza virus (IV), parainfluenza virus (PIV), coronavirus (CoV) and adenovirus (AdV), and highlight potential targets and compounds for therapeutic approaches.


Assuntos
Ciclo do Ácido Cítrico/fisiologia , Metabolismo Energético/fisiologia , Ácidos Graxos/biossíntese , Glicólise/fisiologia , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Adenoviridae/metabolismo , Coronavirus/metabolismo , Humanos , Orthomyxoviridae/metabolismo , Vírus da Parainfluenza 1 Humana/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Rhinovirus/metabolismo
2.
Cells ; 10(8)2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34440666

RESUMO

Adenovirus (Ad) has risen to be a promising alternative to conventional cancer therapy. However, systemic delivery of Ad, which is necessary for the treatment of metastatic cancer, remains a major challenge within the field, owing to poor tumor tropism and nonspecific hepatic tropism of the virus. To address this limitation of Ad, we have synthesized two variants of folic acid (FA)-conjugated methoxy poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]-L-glutamate (P5N2LG-FA and P5N5LG-FA) using 5 kDa poly(ethylene glycol) (PEG) with a different level of protonation (N2 < N5 in terms of charge), along with a P5N5LG control polymer without FA. Our findings demonstrate that P5N5LG, P5N2LG-FA, and P5N5LG-FA exert a lower level of cytotoxicity compared to 25 kDa polyethyleneimine. Furthermore, green fluorescent protein (GFP)-expressing Ad complexed with P5N2LG-FA and P5N5LG-FA (Ad/P5N2LG-FA and Ad/P5N5LG-FA, respectively) exerted superior transduction efficiency compared to naked Ad or Ad complexed with P5N5LG (Ad/P5N5LG) in folate receptor (FR)-overexpressing cancer cells (KB and MCF7). All three nanocomplexes (Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA) internalized into cancer cells through coxsackie adenovirus receptor-independent endocytic mechanism and the cell uptake was more efficient than naked Ad. Importantly, the cell uptake of the two FA functionalized nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) was dependent on the complementary interaction of FA-FR. Systemically administered Ad/P5N5LG, Ad/P5N2LG-FA, and Ad/P5N5LG-FA showed exponentially higher retainment of the virus in blood circulation up to 24 h post-administration compared with naked Ad. Both tumor-targeted nanocomplexes (Ad/P5N2LG-FA and Ad/P5N5LG-FA) showed significantly higher intratumoral accumulation than naked Ad or Ad/P5N5LG via systemic administration. Both tumor-targeted nanocomplexes accumulated at a lower level in liver tissues compared to naked Ad. Notably, the nonspecific accumulation of Ad/P5N2LG-FA was significantly lower than Ad/P5N5LG-FA in several normal organs, while exhibiting a significantly higher intratumoral accumulation level, showing that careful optimization of polyplex surface charge is critical to successful tumor-targeted systemic delivery of Ad nanocomplexes.


Assuntos
Adenoviridae/genética , Materiais Biocompatíveis/química , Vetores Genéticos , Nanopartículas , Neoplasias/genética , Polímeros/química , Transdução Genética , Células A549 , Adenoviridae/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Células MCF-7 , Masculino , Camundongos Nus , Neoplasias/metabolismo , Propriedades de Superfície , Distribuição Tecidual
3.
Biochem Biophys Res Commun ; 548: 211-216, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33647798

RESUMO

In the past decades, remarkable efforts have been made to unravel the regulation of adipose tissue metabolism, given the increasing prevalence of obesity and its huge impact on human health. Wnt signaling pathway is closely involved in this entity. As extracellular inhibitors to Wnt signaling, secreted protein Dickkopfs (Dkks) may be potential targets to combat obesity and related metabolic disorders. In this study, we showed that Dkk2 was a beige fat-enriched adipokine to regulate adipogenesis. Dkk2 was strikingly expressed in beige fat depot compared to classic white, brown, and subcutaneous fat. Dkk2 treatment inhibited adipogenesis in 3T3-L1 pre-adipocytes, C3H10T1/2 mesenchymal stem cells, and primary bone marrow mesenchymal stromal cells. Activation of the master adipogenic factor PPARγ by the synthetic Thiazolidinedione ligand rosiglitazone largely rescued the inhibition of adipogenesis by Dkk2. Furthermore, adenoviral overexpression of Dkk2 in the liver to mimic its gain-of-function showed minimal effect on whole-body metabolism. These results collectively suggest that Dkk2 is a first-in-class beige fat adipokine and functions mainly through a paracrine manner to inhibit adipogenesis rather than as an endocrine factor. Our findings aid a better understanding of beige fat function and regulation and further, provide a potential therapeutic target for treating obesity.


Assuntos
Adipogenia , Adipocinas/metabolismo , Tecido Adiposo Bege/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células 3T3-L1 , Adenoviridae/metabolismo , Adipócitos Bege/efeitos dos fármacos , Adipócitos Bege/metabolismo , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/metabolismo , Rosiglitazona/farmacologia
4.
J Bone Miner Metab ; 39(4): 534-546, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33569722

RESUMO

INTRODUCTION: To investigate the role of LncRNA PVT1 (plasmacytoma variant translocation 1) in hyperglycemia-triggered cartilage damage using the diabetic osteoarthritis (OA) mice model. MATERIALS AND METHODS: Streptozotocin (STZ) was used to induce mouse diabetes. Knee OA model was induced through transection of anterior cruciate ligament (ACLT). Severity of arthritis was assessed histologically by Safranin O-Fast Green Staining using Mankin Scores. LncRNA PVT1 and miR-146a were detected by real-time polymerase chain reaction (PCR) in cartilage tissue. Moreover, the interaction among PVT1, miR-146a, and SMAD4 was examined by luciferase reporter assays. Mice were injected intra-articularly with ad-siRNA-PVT1 and ad-siRNA scramble control. Articular concentrations of TNF-α, IL-1, IL-6 and TGF-ß1 were determined using enzyme-linked immunosorbent assay. Levels of type II Collagen (COL2A1), TGF-ß1, p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4 and nuclear SMAD4 were detected by western blot analysis. RESULTS: PVT1 expression was significantly increased, whereas miR-146a was markedly decreased in diabetic OA mice than in non-diabetic OA and control. Increased PVT1 expression in diabetic OA mice was significantly associated with Mankin score and reduced miR-146a as well as Collagen alpha-1(II) (COL2A1) expressions. In vivo, intra-articular injection of ad-siRNA-PVT1 efficiently increased miR-146a and COL2A1 expressions, alleviated joint inflammation, decreased the expression of pro-inflammatory mediators, and suppressed TGF-ß/SMAD4 pathway in diabetic OA mice. CONCLUSIONS: Our results demonstrate LncRNA PVT1 is involved in cartilage degradation in diabetic OA and correlated with disease severity. Efficiency of ad-siRNA-PVT1 in controlling joint inflammation in diabetic OA mice is associated with the suppression of the expression of miR-146a, pro-inflammatory cytokines and activation of TGF-ß/SMAD4 pathway.


Assuntos
Cartilagem Articular/patologia , Diabetes Mellitus Experimental/genética , Regulação para Baixo , MicroRNAs/genética , Osteoartrite/genética , RNA Longo não Codificante/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adenoviridae/metabolismo , Animais , Sequência de Bases , Cartilagem Articular/metabolismo , Colágeno Tipo II/metabolismo , Regulação para Baixo/genética , Células HEK293 , Humanos , Hiperglicemia/complicações , Hiperglicemia/patologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Osteoartrite/patologia , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
5.
Exp Biol Med (Maywood) ; 246(10): 1228-1238, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33535808

RESUMO

Ras gene mutation or overexpression can lead to tumorigenesis in multiple kinds of cancer, including glioma. However, no drugs targeting Ras or its expression products have been approved for clinical application thus far. Adenoviral gene therapy is a promising method for the treatment of glioma. In this study, the human glioma cell line U251 was co-cultured with recombinant adenovirus KGHV500, and the anti-tumor effects of KGHV500 were determined by MTT, scratch test, Transwell invasion, and apoptosis assays. Then, KGHV500 was delivered via the intravenous injection of CIK cells into glioma xenografts. Tumor volume, ki67 proliferation index, apoptosis levels, and anti-p21Ras scFv expression were tested to evaluate targeting ability, anti-tumor efficacy, and safety. We found that the KGHV500 exhibited anti-tumor activity in U251 cells and increased the intracellular expression of anti-p21Ras scFv compared with that in the control groups. CIK cells delivered KGHV500 to U251 glioma cell xenografts and enhanced anti-tumor activity against glioma xenografts compared to that produced by the control treatment. In conclusion, targeting Ras is a useful therapeutic strategy for gliomas and other Ras-driven cancers, and the delivery of anti-p21Ras scFv by recombinant adenovirus and CIK cells may play an essential role in the therapy of Ras-driven cancers.


Assuntos
Adenoviridae/metabolismo , Células Matadoras Induzidas por Citocinas/metabolismo , Glioma/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Anticorpos de Cadeia Única/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Recombinação Genética/genética , Proteínas Virais/metabolismo
6.
J Cell Physiol ; 236(9): 6559-6570, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33634483

RESUMO

Acute liver injury (ALI) is a rapid pathological process that may cause severe liver disease and may even be life-threatening. During ALI, the function of males absent on the first (MOF) has not yet been elucidated. In this study, we unveiled the expression pattern of MOF during carbon tetrachloride (CCl4 )-induced ALI and role of MOF in the regulation of liver regeneration. In the process of ALI, MOF is significantly overexpressed in the liver injury area. Knockdown of Mof attenuated CCl4 -induced ALI, and promoted liver cell proliferation, hepatic stellate cell activation and aggregation to the injured area, and liver fibrosis. Simultaneously, overexpression of Mof aggravated liver dysfunction caused by ALI. By directly binding to the promoter, MOF suppressed the transcriptional activation of Igf1. Knockdown of Mof promotes the expression of Igf1 and activates the Insulin-like growth factor 1 signaling pathway in the liver. Through this pathway, Knockdown of Mof reduces CCl4 -induced ALI and promotes liver regeneration. Our results provide the first demonstration for MOF contributing to ALI. Further understanding of the role of MOF in ALI may lead to new therapeutic strategies for ALI.


Assuntos
Histona Acetiltransferases/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fígado/lesões , Fígado/metabolismo , Ativação Transcricional/genética , Doença Aguda , Adenoviridae/metabolismo , Animais , Tetracloreto de Carbono , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Imidazóis/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Regeneração Hepática/efeitos dos fármacos , Regeneração Hepática/genética , Masculino , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos
7.
Proc Natl Acad Sci U S A ; 118(5)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33514660

RESUMO

An effective vaccine that can protect against HIV infection does not exist. A major reason why a vaccine is not available is the high mutability of the virus, which enables it to evolve mutations that can evade human immune responses. This challenge is exacerbated by the ability of the virus to evolve compensatory mutations that can partially restore the fitness cost of immune-evading mutations. Based on the fitness landscapes of HIV proteins that account for the effects of coupled mutations, we designed a single long peptide immunogen comprising parts of the HIV proteome wherein mutations are likely to be deleterious regardless of the sequence of the rest of the viral protein. This immunogen was then stably expressed in adenovirus vectors that are currently in clinical development. Macaques immunized with these vaccine constructs exhibited T-cell responses that were comparable in magnitude to animals immunized with adenovirus vectors with whole HIV protein inserts. Moreover, the T-cell responses in immunized macaques strongly targeted regions contained in our immunogen. These results suggest that further studies aimed toward using our vaccine construct for HIV prophylaxis and cure are warranted.


Assuntos
Vacinas contra a AIDS/imunologia , Adenoviridae/metabolismo , Vetores Genéticos/metabolismo , HIV-1/imunologia , Proteoma/metabolismo , Sequência de Aminoácidos , Animais , Antígenos Virais/imunologia , Feminino , Infecções por HIV/imunologia , Imunização , Macaca mulatta , Masculino , Linfócitos T Citotóxicos/imunologia , Proteínas Virais/química , Proteínas Virais/metabolismo
8.
Sci Rep ; 11(1): 856, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441685

RESUMO

Adequate viral replication in tumor cells is the key to improving the anti-cancer effects of oncolytic adenovirus therapy. In this study, we introduced short hairpin RNAs against death-domain associated protein (Daxx), a repressor of adenoviral replication, and precursor terminal protein (pTP), an initiator of adenoviral genome replication, into adenoviral constructs to determine their contributions to viral replication. Both Daxx downregulation and pTP overexpression increased viral production in variety of human cancer cell lines, and the enhanced production of virus progeny resulted in more cell lysis in vitro, and tumor regression in vivo. We confirmed that increased virus production by Daxx silencing, or pTP overexpression, occurred using different mechanisms by analyzing levels of adenoviral protein expression and virus production. Specifically, Daxx downregulation promoted both virus replication and oncolysis in a consecutive manner by optimizing IVa2-based packaging efficiency, while pTP overexpression by increasing both infectious and total virus particles but their contribution to increased viral production may have been damaged to some extent by their another contribution to apoptosis and autophagy. Therefore, introducing both Daxx shRNA and pTP in virotherapy may be a suitable strategy to increase apoptotic tumor-cell death and to overcome poor viral replication, leading to meaningful reductions in tumor growth in vivo.


Assuntos
Proteínas Correpressoras/metabolismo , Chaperonas Moleculares/metabolismo , Terapia Viral Oncolítica/métodos , Replicação Viral/fisiologia , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/fisiologia , Proteínas E2 de Adenovirus/metabolismo , Proteínas E2 de Adenovirus/fisiologia , Linhagem Celular Tumoral , Proteínas Correpressoras/fisiologia , Humanos , Chaperonas Moleculares/fisiologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/metabolismo , RNA Interferente Pequeno/genética , Proteínas Virais/genética , Replicação Viral/genética
9.
Cells ; 10(2)2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33513935

RESUMO

Oncolytic viruses provide a biologically multi-faceted treatment option for patients who cannot be cured with currently available treatment options. We constructed an oncolytic adenovirus, TILT-123, to support T-cell therapies and immune checkpoint inhibitors in solid tumors. Adenoviruses are immunogenic by nature, are easy to produce in large quantities, and can carry relatively large transgenes. They are the most commonly used gene therapy vectors and are well tolerated in patients. TILT-123 expresses two potent cytokines, tumor necrosis factor alpha and interleukin-2, to stimulate especially the T-cell compartment in the tumor microenvironment. Before entering clinical studies, the safety and biodistribution of TILT-123 was studied in Syrian hamsters and in mice. The results show that TILT-123 is safe in animals as monotherapy and in combination with an immune checkpoint inhibitor anti-PD-1. The virus treatment induces acute changes in circulating immune cell compartments, but the levels return to normal by the middle of the treatment period. The virus is rapidly cleared from healthy tissues, and it does not cause damage to vital organs. The results support the initiation of a phase 1 dose-escalation trial, where melanoma patients receiving a tumor-infiltrating lymphocyte therapy are treated with TILT-123 (NCT04217473).


Assuntos
Adenoviridae/metabolismo , Citocinas/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Vírus Oncolíticos/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos Neutralizantes/metabolismo , Linhagem Celular Tumoral , Cricetinae , Feminino , Injeções , Masculino , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Especificidade de Órgãos , Receptor de Morte Celular Programada 1/metabolismo , Distribuição Tecidual , Transgenes , Replicação Viral/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Cell Mol Med ; 25(1): 27-36, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33128338

RESUMO

The effects of long-term nitrate therapy are compromised due to protein S-Nitrosylation, which is mediated by nitric oxide (NO). This study is to determine the role of Akt S-Nitrosylation in the recovery of heart functions after ischaemia. In recombinant Akt protein and in HEK293 cells, NO donor decreased Akt activity and induced Akt S-Nitrosylation, but was abolished if Akt protein was mutated by replacing cysteine 296/344 with alanine (Akt-C296/344A). In endothelial cells, NO induced Akt S-Nitrosylation, reduced Akt activity and damaged multiple cellular functions including proliferation, migration and tube formation. These alterations were ablated if cells expressed Akt-C296/344A mutant. In Apoe-/- mice, nitroglycerine infusion increased both Akt S-Nitrosylation and infarct size, reduced Akt activity and capillary density, and delayed the recovery of cardiac function in ischaemic hearts, compared with mice infused with vehicle. Importantly, these in vivo effects of nitroglycerine in Apoe-/- mice were remarkably prevented by adenovirus-mediated enforced expression of Akt-C296/344A mutant. In conclusion, long-term usage of organic nitrate may inactivate Akt to delay ischaemia-induced revascularization and the recovery of cardiac function through NO-mediated S-Nitrosylation.


Assuntos
Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Revascularização Miocárdica , Nitratos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adenoviridae/metabolismo , Sequência de Aminoácidos , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisteína/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Mutação/genética , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/metabolismo , Nitroglicerina/farmacologia , Nitroprussiato/farmacologia , Nitrosação
11.
Am J Respir Cell Mol Biol ; 64(1): 79-88, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32991819

RESUMO

Preclinical mouse models that recapitulate some characteristics of coronavirus disease (COVID-19) will facilitate focused study of pathogenesis and virus-host responses. Human agniotensin-converting enzyme 2 (hACE2) serves as an entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect people via binding to envelope spike proteins. Herein we report development and characterization of a rapidly deployable COVID-19 mouse model. C57BL/6J (B6) mice expressing hACE2 in the lung were transduced by oropharyngeal delivery of the recombinant human adenovirus type 5 that expresses hACE2 (Ad5-hACE2). Mice were infected with SARS-CoV-2 at Day 4 after transduction and developed interstitial pneumonia associated with perivascular inflammation, accompanied by significantly higher viral load in lungs at Days 3, 6, and 12 after infection compared with Ad5-empty control group. SARS-CoV-2 was detected in pneumocytes in alveolar septa. Transcriptomic analysis of lungs demonstrated that the infected Ad5-hACE mice had a significant increase in IFN-dependent chemokines Cxcl9 and Cxcl10, and genes associated with effector T-cell populations including Cd3 g, Cd8a, and Gzmb. Pathway analysis showed that several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched in the data set, including cytokine-cytokine receptor interaction, the chemokine signaling pathway, the NOD-like receptor signaling pathway, the measles pathway, and the IL-17 signaling pathway. This response is correlative to clinical response in lungs of patients with COVID-19. These results demonstrate that expression of hACE2 via adenovirus delivery system sensitized the mouse to SARS-CoV-2 infection and resulted in the development of a mild COVID-19 phenotype, highlighting the immune and inflammatory host responses to SARS-CoV-2 infection. This rapidly deployable COVID-19 mouse model is useful for preclinical and pathogenesis studies of COVID-19.


Assuntos
Células Epiteliais Alveolares/imunologia , COVID-19/imunologia , Expressão Gênica , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Adenoviridae/genética , Adenoviridae/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2/biossíntese , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Animais , COVID-19/genética , COVID-19/metabolismo , COVID-19/patologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transdução de Sinais/genética , Transdução Genética
12.
Life Sci ; 265: 118762, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33189825

RESUMO

AIMS: This study is to investigate the role of adenovirus type 36 (Ad36) in inducing differentiation of human adipose-derived stem cells (hADSCs) into brown adipocytes. MAIN METHODS: The hADSCs were induced to differentiate into adipocytes by a cocktail method and Ad36, respectively. They were collected on the 2nd, 4th, 6th, and 8th day, respectively. LncRNA ROR was silenced by siRNA. RT-qPCR and Western-blot were used to detect the mRNA and protein levels. Transmission electron microscopy was used to observe the mitochondria. KEY FINDINGS: The mRNA and protein expression levels of LncRNA ROR, Cidea, Dio2, Fgf21, Ucp1, Prdm16, Cox5b, Atp5o, Atp6, and Nd2 in the Ad36 induction group were significantly higher than those in the cocktail induction group. The expression levels of Leptin mRNA and protein in the Ad36 induction group were significantly lower than those in the cocktail induction group. After siRNA knockdown of LncRNA ROR, mRNA and protein expression levels of Cidea, Dio2, Fgf21, Ucp1, Prdm16, Cox5b, Atp5o, Atp6 and Nd2 were significantly lower than the control group during the induction of hADSC differentiation into adipocytes by Ad36. Additionally, mitochondria in the Ad36 induction group was increased compared to that in the cocktail induction group. SIGNIFICANCE: Ad36 may promote the differentiation of hADSCs into brown adipocytes by up-regulating LncRNA ROR.


Assuntos
Adenoviridae/metabolismo , Infecções por Adenovirus Humanos/metabolismo , Adipócitos Marrons/virologia , Células-Tronco Mesenquimais/metabolismo , RNA Longo não Codificante/metabolismo , Adipócitos Marrons/metabolismo , Adipócitos Marrons/fisiologia , Adipócitos Marrons/ultraestrutura , Western Blotting , Diferenciação Celular , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
13.
Biomed Pharmacother ; 134: 110932, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33370632

RESUMO

Oncolytic viruses have attracted attention as a promising strategy in cancer therapy owing to their ability to selectively infect and kill tumor cells, without affecting healthy cells. They also exert their anti-tumor effects by releasing immunostimulatory molecules from dying cancer cells. Several regulatory mechanisms, such as autophagy, contribute to the anti-tumor properties of oncolytic viruses. Autophagy is a conserved catabolic process in responses to various stresses, such as nutrient deprivation, hypoxia, and infection that produces energy by lysosomal degradation of intracellular contents. Autophagy can support infectivity and replication of the oncolytic virus and enhance their anti-tumor effects via mediating oncolysis, autophagic cell death, and immunogenic cell death. On the other hand, autophagy can reduce the cytotoxicity of oncolytic viruses by providing survival nutrients for tumor cells. In his review, we summarize various types of oncolytic viruses in clinical trials, their mechanism of action, and autophagy machinery. Furthermore, we precisely discuss the interaction between oncolytic viruses and autophagy in cancer therapy and their combinational effects on tumor cells.


Assuntos
Autofagia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/metabolismo , Adenoviridae/metabolismo , Animais , Morte Celular Autofágica , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Humanos , Morte Celular Imunogênica , Vírus do Sarampo/metabolismo , Camundongos , Neoplasias/metabolismo , Simplexvirus/metabolismo , Vesiculovirus/metabolismo , Replicação Viral
14.
Biomolecules ; 10(11)2020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-33187345

RESUMO

Direct interaction between intrinsically disordered proteins (IDPs) is often difficult to characterize hampering the elucidation of their binding mechanism. Particularly challenging is the study of fuzzy complexes, in which the intrinsically disordered proteins or regions retain conformational freedom within the assembly. To date, nuclear magnetic resonance spectroscopy has proven to be one of the most powerful techniques to characterize at the atomic level intrinsically disordered proteins and their interactions, including those cases where the formed complexes are highly dynamic. Here, we present the characterization of the interaction between a viral protein, the Early region 1A protein from Adenovirus (E1A), and a disordered region of the human CREB-binding protein, namely the fourth intrinsically disordered linker CBP-ID4. E1A was widely studied as a prototypical viral oncogene. Its interaction with two folded domains of CBP was mapped, providing hints for understanding some functional aspects of the interaction with this transcriptional coactivator. However, the role of the flexible linker connecting these two globular domains of CBP in this interaction was never explored before.


Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteína de Ligação a CREB/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Adenoviridae/genética , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Proteínas E1A de Adenovirus/genética , Proteína de Ligação a CREB/genética , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Ligação Proteica , Domínios Proteicos
15.
PLoS One ; 15(11): e0242725, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33253191

RESUMO

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites.


Assuntos
Adenoviridae/química , Íntrons , Regiões Promotoras Genéticas , Sítios de Splice de RNA , Fatores de Processamento de RNA/química , RNA Viral/química , Proteínas Repressoras/química , Adenoviridae/metabolismo , Cristalografia por Raios X , Humanos , Fatores de Processamento de RNA/metabolismo , RNA Viral/metabolismo , Proteínas Repressoras/metabolismo
16.
Int J Mol Sci ; 21(19)2020 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-32998359

RESUMO

Mitogen-activated protein kinase phosphatase-5 (MKP-5) is a regulator of extracellular signaling that is known to regulate lipid metabolism. In this study, we found that obesity caused by a high-fat diet (HFD) decreased the expression of MKP-5 in the pancreas and primary islet cells derived from mice. Then, we further investigated the role of MKP-5 in the protection of islet cells from lipotoxicity by modulating MKP-5 expression. As a critical inducer of lipotoxicity, palmitic acid (PA) was used to treat islet ß-cells. We found that MKP-5 overexpression restored PA-mediated autophagy inhibition in Rin-m5f cells and protected these cells from PA-induced apoptosis and dysfunction. Consistently, a lack of MKP-5 aggravated the adverse effects of lipotoxicity. Islet cells from HFD-fed mice were infected using recombinant adenovirus expressing MKP-5 (Ad-MKP-5), and we found that Ad-MKP-5 was able to alleviate HFD-induced apoptotic protein activation and relieve the HFD-mediated inhibition of functional proteins. Notably, HFD-mediated impairments in autophagic flux were restored by Ad-MKP-5 transduction. Furthermore, the autophagy inhibitor 3-methyladenine (3-MA) was used to treat Rin-m5f cells, confirming that the MKP-5 overexpression suppressed apoptosis, dysfunction, inflammatory response, and oxidative stress induced by PA via improving autophagic signaling. Lastly, employing c-Jun amino-terminal kinas (JNK), P38, or extracellular-regulated kinase (ERK) inhibitors, we established that the JNK and P38 MAPK pathways were involved in the MKP-5-mediated apoptosis, dysfunction, and autophagic inhibition observed in islet ß cells in response to lipotoxicity.


Assuntos
Autofagia/genética , Fosfatases de Especificidade Dupla/genética , Ilhotas Pancreáticas/enzimologia , Metabolismo dos Lipídeos/genética , Obesidade/genética , Adenina/análogos & derivados , Adenina/farmacologia , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Fosfatases de Especificidade Dupla/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Obesidade/enzimologia , Obesidade/etiologia , Obesidade/patologia , Ácido Palmítico/antagonistas & inibidores , Ácido Palmítico/toxicidade , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Transdução Genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Viruses ; 12(10)2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33050291

RESUMO

Superimposition of protein structures is key in unravelling structural homology across proteins whose sequence similarity is lost. Structural comparison provides insights into protein function and evolution. Here, we review some of the original findings and thoughts that have led to the current established structure-based phylogeny of viruses: starting from the original observation that the major capsid proteins of plant and animal viruses possess similar folds, to the idea that each virus has an innate "self". This latter idea fueled the conceptualization of the PRD1-adenovirus lineage whose members possess a major capsid protein (innate "self") with a double jelly roll fold. Based on this approach, long-range viral evolutionary relationships can be detected allowing the virosphere to be classified in four structure-based lineages. However, this process is not without its challenges or limitations. As an example of these hurdles, we finally touch on the difficulty of establishing structural "self" traits for enveloped viruses showcasing the coronaviruses but also the power of structure-based analysis in the understanding of emerging viruses.


Assuntos
Adenoviridae/metabolismo , Proteínas do Capsídeo/metabolismo , Coronavirus/metabolismo , Estrutura Terciária de Proteína/fisiologia , Rhinovirus/metabolismo , Adenoviridae/genética , Coronavirus/genética , Cristalografia por Raios X , Genoma Viral/genética , Rhinovirus/genética , Estruturas Virais/metabolismo
19.
BMB Rep ; 53(11): 565-575, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32958121

RESUMO

Gene therapy is emerging as a treatment option for inherited genetic diseases. The success of this treatment approach greatly depends upon gene delivery vectors. Researchers have attempted to harness the potential of viral vectors for gene therapy applications over many decades. Among the viral vectors available, gutless adenovirus (GLAd) has been recognized as one of the most promising vectors for in vivo gene delivery. GLAd is constructed by deleting all the viral genes from an adenovirus. Owing to this structural feature, the production of GLAd requires a helper that supplies viral proteins in trans. Conventionally, the helper is an adenovirus. Although the helper adenovirus efficiently provides helper functions, it remains as an unavoidable contaminant and also generates replicationcompetent adenovirus (RCA) during the production of GLAd. These two undesirable contaminants have raised safety concerns and hindered the clinical applications of GLAd. Recently, we developed helper virus-free gutless adenovirus (HF-GLAd), a new version of GLAd, which is produced by a helper plasmid instead of a helper adenovirus. Utilization of this helper plasmid eliminated the helper adenovirus and RCA contamination in the production of GLAd. HF-GLAd, devoid of helper adenovirus and RCA contaminants, will facilitate its clinical applications. In this review, we discuss the characteristics of adenoviruses, the evolution and production of adenoviral vectors, and the unique features of HF-GLAd as a new platform for gene therapy. Furthermore, we highlight the potential applications of HF-GLAd as a gene delivery vector for the treatment of various inherited genetic diseases. [BMB Reports 2020; 53(11): 565-575].


Assuntos
Adenoviridae/genética , Adenoviridae/metabolismo , Terapia Genética/métodos , Linhagem Celular , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Vírus Auxiliares/genética , Vírus Auxiliares/metabolismo , Humanos , Integrases/genética , Plasmídeos/genética , Proteínas Virais/genética
20.
Exp Cell Res ; 396(1): 112185, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32828827

RESUMO

BACKGROUND: Apoptin can specifically kill cancer cells but has no toxicity to normal cells. Human telomerase reverse transcriptase (hTERT) acts as a tumor-specific promoter, triggering certain genes to replicate or express only in tumor cells, conferring specific replication and killing abilities. This study aimed at investigating the anticancer potential of the recombinant adenovirus Ad-apoptin-hTERTp-E1a (Ad-VT) in ovarian cancer treatment. METHODS: Crystal Violet staining and WST-1 assays were used to analyze the inhibitory effect of Ad-VT on ovarian cancer SKOV3 and OVCAR-3 cells. Ad-VT-induced apoptosis of ovarian cancer cells, was detected using Hoechst, Annexin V-FITC/PI, JC-1 staining. Cell migration and invasion of ovarian cancer cells were detected using cell-scratch and Transwell assays. The pGL4.51 plasmid was used to transfect and to generate SKOV3-LUC cells, that stably express luciferase. The in vivo tumor inhibition effect of Ad-VT was subsequently confirmed using a tumor-bearing nude mouse model. RESULTS: Ad-VT had a strong apoptosis-inducing effect on SKOV3 and OVCAR-3 cells, that was mainly mediated through the mitochondrial apoptotic pathway. The Ad-VT could significantly increase the inhibition of ovarian cancer cell migration and invasion. The Ad-VT also can inhibit tumor growth and reduce toxicity in vivo. CONCLUSIONS: The recombinant adenovirus, comprising the apoptin protein and the hTERTp promoter, was able to inhibit the growth of ovarian cancer cells and promote their apoptosis.


Assuntos
Adenoviridae/genética , Carcinoma Epitelial do Ovário/genética , Vírus da Anemia da Galinha/genética , Terapia Viral Oncolítica/métodos , Neoplasias Ovarianas/genética , Proteínas Virais/genética , Adenoviridae/metabolismo , Animais , Apoptose/genética , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/mortalidade , Carcinoma Epitelial do Ovário/virologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Vírus da Anemia da Galinha/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/virologia , Análise de Sobrevida , Transgenes , Carga Tumoral , Proteínas Virais/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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